Starmaker Exhibits Properties of an Intrinsically Disordered Protein

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Biomacromolecules 2008, 9, 2118–2125

Starmaker Exhibits Properties of an Intrinsically Disordered Protein Tomasz M. Kapłon,† Grzegorz Rymarczyk,† Małgorzata Nocula-Ługowska,† Michał Jako´b,† Marian Kochman,† Marek Lisowski,‡ Zbigniew Szewczuk,‡ and Andrzej Oz˙yhar*,† Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzez˙e Wyspian´skiego 27, 50-370 Wrocław, Poland, and Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wroclaw, Poland Received February 8, 2008; Revised Manuscript Received May 6, 2008

Fish otoliths composed of calcium carbonate and an organic matrix play a primary role in gravity sensing and the perception of sound. Starmaker (Stm) was the first protein found to be capable of influencing the process of biomineralization of otoliths. Stm dictates the shape, size, and selection of calcium carbonate polymorphs in a concentration-dependent manner. To facilitate exploration of the molecular basis of Stm function, we have developed and optimized a protocol for efficient expression and purification of the homogeneous nontagged Stm. The homogeneous nontagged Stm corresponds to its functional form, which is devoid of a signal peptide. A comprehensive biochemical and biophysical analysis of recombinant Stm, along with in silico examinations, indicate for the first time that Stm exhibits the properties of intrinsically disordered proteins. The functional significance of Stm having intrinsically disordered protein properties and its possible role in controlling the formation of otoliths is discussed.

Introduction Otoliths in bony fishes and otoconia in mammals are composite crystals consisting of calcium carbonate and an organic fraction composed mostly of proteoglycans and glycoproteins. These biominerals are part of the gravity and linear acceleration detection system of the inner ear. They are involved in the perception of balance, as well as in the reception of sound.1 Several human disorders are associated with otoconia deficiency, otoconia abnormality, or age-related otoconia degeneration.2,3 Although otoliths and otoconia are indispensable for proper postural function and orientation in the environment, only limited knowledge has been available to date about the molecular mechanisms involved in the biogenesis of these composite crystals. In zebrafish (Danio rerio) the biomineralization of otoliths was shown to be strictly controlled by a Starmaker protein (Stm). Stm was the first protein found to be capable of influencing the process of biomineralization. Using RNA antisense technology, it has been shown that Stm acts as a crystal growth inhibitor regulating the growth, shape, and crystal lattice in a concentration-dependent manner.4 With respect to these features, Stm is akin to human Dentin sialophosphoprotein (DSPP), a key factor required for proper teeth mineralization and also expressed in the human ear.2,3 Both Stm and DSPP seem to have analogous functions despite their relatively low sequence homology.4,5 The expression of Stm is restricted to cells from the sensory epithelium, including neuroepithelial patches within the inner ear and clusters of the sensory hair cell epithelium of the lateral line organ. Stm was also found in the pineal gland.4 Because otoliths are extracellularly formed in the lumen of the otic cavity, all elements necessary for their formation need to be secreted to this compartment. In view of this fact, the computationally predicted * To whom correspondence should be addressed. Phone: 004871 320 63 33. Fax: 004871 320 63 37. E-mail: [email protected]. † Wrocław University of Technology. ‡ University of Wrocław.

signal peptide of Stm was postulated to be active within Stm, and Otopetrin 1, a transmembrane protein located in the hair cells of neuroepithelium, was suggested to be involved in the secretion of Stm.6 Further analysis of Stm amino acid sequence demonstrated some additional structural elements like four highly conserved 13-amino acid repeats, the function of which is still unknown, and a 60 amino acid-long region rich in alternating D and S residues.4 A similar region rich in S and D residues arranged in a specific pattern, defined as a repeating DSS motif, was found in DSPP. As was postulated by George,7 such an arrangement, combined with the phosphorylation of abundant S residues, could create a spatial structure capable of binding high amounts of calcium ions, which is probably essential for bridging inorganic crystals and the proteinaceous skeletons of biominerals. Despite the identification of Stm and other proteins involved in the formation of otoliths,8 the molecular basis of their function in biomineralization remains unknown, possibly due to the difficulty of obtaining pure preparations of these proteins, resulting from the their low concentration in natural sources. To further detail biochemical studies aimed at understanding mechanisms by which Stm acts as a key factor regulating otolith formation, we have developed and optimized a protocol for the efficient expression and purification of Stm. A comprehensive biochemical and biophysical analysis along with in silico examinations importantly indicate for the first time that Stm exhibits features typical of intrinsically disordered proteins. The functional significance of this structural feature of Stm has been analyzed and its possible role in the mineralization of otoliths is discussed.

Materials and Methods Buffers. All buffers were prepared at 24 °C. Buffer A was 50 mM Na2HPO4, 150 mM NaCl, 10% (v/v) glycerol, 1 mM 2-mercaptoethanol, pH 7.0. Buffer B was 10 mM Tris, 100 mM NaCl, 10% (v/v) glycerol, pH 7.0. Buffer L was 10 mM Na2HPO4, 2 mM KH2PO4,

10.1021/bm800135m CCC: $40.75  2008 American Chemical Society Published on Web 07/18/2008

Starmaker Exhibits Properties of Disordered Protein 137 mM NaCl, 6 mM (NH4)2SO4, 3 mM KCl, 1 mM 2-mercaptoethanol, pH 7.0. Construction of Expression Vectors. Full-length Stm cDNA4 was amplified using a PCR9 and the following primers 5′-GCCCGCGGATCCctgtcccggacagtgtttg-3′ (forward) and 5′-GCCCGCAAGCTTggaaatcggcatagaagttttg-3′ (reverse). To amplify Stm cDNA devoid of the signal peptide (SP) coding sequence the forward and reverse primers were 5′-GCCCGCGGATCCgcaccagtgagcaataacaatg-3′ and 5′-GCCCGCAAGCTTggaaatcggcatagaagttttg-3′, respectively. Lowercase letters in primer sequences represent sequences originating from the Stm gene, whereas uppercase letters represent nucleotides added for cloning purposes. Each primer introduced a specific endonuclease recognition site (underlined sequences for BamHI and for HindIII), enabling subsequent insertion of amplified fragments into linearized plasmid. PCR products were purified using a NucleoTraPCR kit (MachereyNagel, Germany), digested by BamHI and HindIII endonucleases and subcloned into a set of pQE-80L vector (Qiagen, Germany) derivatives obtained in our laboratory (not shown). Each of the constructed plasmids encoded one of the four recombinant proteins (Stm-His, HisStm∆SP, Strep-Stm∆SP-His, and Stm∆SP) schematically depicted in Figure 1A. The presence of the inserts within each construct was confirmed by restriction analysis (data not shown) and the purified constructs were verified by sequencing, using a CycleReader Auto DNA Sequencing Kit (MBI Fermentas, Lithuania) and the Pharmacia Biotech ALF express analyzer. Expression Analysis. Prior to elaboration of the purification procedure the expression of each recombinant Stm variant was evaluated. Briefly, BL21(DE3) or BL21(DE3)pLysS E. coli cells (Novagen, Germany) were transformed with 2 ng of the respective recombinant pQE-80L derivative that encoded the desired Stm protein (Figure 1A) and plated on LB-agar plates, containing appropriate antibiotic (35 µg/mL chloramphenicol and/or 50 µg/mL carbenicillin). E. coli cells BL21(DE3) (Novagen, Germany) and carbenicillin at 50 µg/mL were used for Stm-His. After incubation for 16 h at 37 °C, one individual colony was picked for each construct and used to inoculate 10 mL of LB medium in a 100 mL shaker flask. After overnight incubation (29 °C, 182 rpm), each of the starting cultures was used to inoculate 100 mL of LB media in a 500 mL Erlenmeyer flask that was placed in a rotary shaker operated at 182 rpm at 29 °C. At an OD600 of 0.60, 1 mL samples were collected, centrifuged for 2 min at 14100 × g, and the resulting bacterial pellets were resuspended in 75 µL of the 2 × SDS sample buffer (124 mM Tris-HCl, 4% (w/v) SDS, 10% (v/v) 2-mercaptoethanol, 20% (v/v) glycerol, 0.005% (w/v) bromphenol blue, pH 6.8)10 and stored at -80 °C. Because the pQE-80L expression vector contains the phage T5 promoter and two lac operator sequences isopropyl-β-thiogalactopyranoside (IPTG), a synthetic analog of lactose was then added to the rest of each culture to a final concentration of 0.25 mM. Then recombinant protein synthesis was carried out for 3 h at 29 °C, 182 rpm. Next, 1 mL culture samples were collected and prepared, as described above, and stored at -80 °C for subsequent SDS/polyacrylamide gel electrophoresis analysis (see below). Purification of Stm∆SP and Strep-Stm∆SP-His. Glycerol stock cultures of host cells containing respective expression vectors were used to inoculate 130 mL of LB medium containing chloramphenicol (35 µg/mL) and carbenicillin (50 µg/mL). After overnight incubation at 29 °C and 182 rpm the starting culture was used to inoculate 4 L of LB medium, and the resulting suspension was divided into thirteen 1 L Erlenmeyer flasks and placed in a rotary shaker operated at 29 °C and 182 rpm. When OD600 of the cultures reached a value of 0.6, synthesis of the recombinant protein was induced by the addition of IPTG to a final concentration of 0.25 mM. After 3 h of incubation, bacterial cells were collected into four 500 mL centrifugal tubes with 10 min of centrifugation at 10000 × g, 4 °C. The resulting sediment was washed with 12 mL of buffer L (for each tube), centrifuged for 10 min at 10000 × g, 4 °C, and finally resuspended in 24 mL of buffer L in a 50 mL Falcon tube and stored at -80 °C.

Biomacromolecules, Vol. 9, No. 8, 2008

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Figure 1. Recombinant derivatives of Stm. (A) The schematic representation of Stm derivatives used in this study. Stm, wild-type full-length Stm containing the signal peptide4 (SP) sequence and consisting of 613 amino acid residues; Stm-His, full-length Stm C-terminally tagged with an affinity tag consisting of the sequence KLHHHHHHHH; His-Stm∆SP, Stm devoid of SP, where ∆SP means deletion of the SP sequence, and N-terminally tagged with an affinity tag consisting of the sequence MRGSHHHHHHGS; Strep-Stm∆SPHis, Stm devoid of SP, and C-terminally tagged with the sequence KLHHHHHHHH and N-terminally with the Strep-tag II peptide WSHPQFEK21 by a GAGS spacer and, simultaneously, MAS residues were placed at the Strep-tag II N-terminus; Stm∆SP, Stm devoid of SP and containing three vector-derived amino acid residues added at the N-terminus (MGS) and C-terminus (KLN). (B) Representative SDS/polyacrylamide gel electrophoresis analysis of Stm derivatives expression. Whole cell extracts obtained from the bacterial expression of the Stm derivatives indicated at the top of the gel were electrophoresed on a 12% SDS/polyacrylamide gel and stained with StainsAll dye as described in Materials and Methods. Lane 1, molecular mass standards; lanes 2, 4, 6, and 8, bacterial cell extracts before induction (NI) of the synthesis of the appropriate Stm derivative; lanes 3, 5, 7, and 9, bacterial cell extracts 3 h after induction (3 h) with the IPTG of synthesis; the arrow indicates that recombinants of Stm are stained in blue.

A total of two pellets of frozen cells, obtained as described above from a total of 2 L of culture, were thawed in a 24 °C water bath and placed on ice. A single freezing and thawing step was sufficient for the bacterial strain resident T7 lysozyme, provided by pLysS plasmid,11 to lyse the cells efficiently. Then DNase I and RNase A were added to the final concentration of 10 µg/mL of each enzyme and the resulting suspension was incubated on ice until there was a loss of viscosity. Proteolytic activity was partially inhibited by the addition of phenylmethylsulfonyl fluoride (PMSF) to the final concentration of 0.5 mg/ mL. The cell extract was subsequently clarified with a 30 min centrifugation at 18500 × g, 4 °C, and the supernatant (45 mL) was fractionated on ice with solid (NH4)2SO4. Proteins from the 55-75%

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(NH4)2SO4 saturation fraction were dissolved in 5 mL of buffer A and dialyzed extensively against buffer A. The resulting solution was concentrated to a volume of 1 mL using an Amicon Ultra-4 centrifugal filter unit (Millipore, Poland) and applied to the HiLoad 16/60 Superdex 200 pg (Amersham Biosciences) column equilibrated with buffer A. ¨ KTAexplorer (Amersham Biosciences) The column was run on the A system at a flow rate of 0.5 mL/min at room temperature. Fractions containing Stm were combined and dialyzed against buffer B. A total of 1 mL of Bio-Gel DNA-GRADE hydroxyapatite resin (HTP; BioRad Laboratories, Germany), incubated overnight at 4 °C in buffer B containing 1 M CaCl2, was packed into a 1 mL HR5/5 column ¨ KTAexplorer system, and (Amersham Biosciences), connected to the A equilibrated with buffer B. Then 10 mL of the preparation obtained from the HiLoad 16/60 Superdex 200 pg column was applied to the HTP column and unbound proteins were washed out with 5 mL of buffer B. We experimentally elaborated the step gradient of 0 mM to 300 mM phosphate, which was used to elute bound proteins at a flow rate of 0.25 mL/min. Fractions containing pure Stm derivatives were combined, concentrated using an Amicon Ultra-4 centrifugal filter unit (Millipore, Poland) and stored at -80 °C. The concentration of purified proteins was determined using the biuret method.12 ESI Mass Spectrometry. Purified Stm∆SP and Strep-Stm∆SP-His were dialyzed against 1% acetic acid and concentrated to 1 mg/mL using an Amicon Ultra-4 centrifugal filter unit (Millipore, Poland). High-resolution mass spectra were obtained on a Bruker Microtof-Q spectrometer (Bruker Daltonik, Germany), equipped with an Apollo II electrospray ionization source with an ion funnel. The protein solution was infused at a flow rate of 3 µL/min. The mass spectrometer was operated in the positive ion mode. The mass resolution was 15000 fwhm. The instrument parameters were as follows: a scan range of m/z 400-2300; dry gas, nitrogen; and temperature, 180 °C. Before performing each measurement the instrument was calibrated externally with the Tunemix mixture (Bruker Daltonik, Germany) in the quadratic regression mode. The acquisition of data was performed using micrOTOFcontrol 2.0 software and data analysis was performed using DataAnalysis software from Daltonik GmbH (Bremen, Germany). Analytical Gel Filtration. The amounts (indicated in the legend to Figure 4) of the purified Stm∆SP were loaded with a total volume of 100 µL onto a Superdex 200 10/300 GL column (Amersham Bio¨ KTAexplorer system and equilibrated with sciences), connected to the A buffer A at a flow rate of 0.5 mL/min. The column was calibrated using the following standard proteins: thyroglobulin (85 Å), apoferritin (67 Å), catalase (52 Å), bovine serum albumin (35.5 Å), ovalbumin (30.5 Å), chymotrypsinogen (20.9 Å), mioglobin (20.2 Å), and cytochrome c (17 Å). The elution volume of each protein was used to calculate KAV factors,13 which were then plotted against corresponding Stokes radii (Figure 4, inset). Calculated KAV was then fitted to the standard curve, and the Stokes radius of Stm∆SP was estimated.13 Circular Dichroism Spectra. Circular dichroism (CD) spectra were recorded using a Jasco J-720 spectropolarimeter. The final spectra resulted from averaging five measurements performed at a temperature of 20 °C, a scanning speed of 20 nm/min, data resolution of 0.2 nm and a bandwidth of 1.0 nm. A 2.15 µM solution of Stm∆SP was used for each measurement performed in 0.1 cm path length quartz cuvette. The contribution of buffer B was subtracted from the original data, which were then smoothed and converted to molar ellipticity. Denaturing conditions were obtained by the addition of an appropriate amount of 6 M GdmCl to the Stm∆SP solution and subsequent incubation at room temperature for 2 h. Fluorescence Measurements. Fluorescence measurements were performed using a FLUOROLOG-3 fluorometer (Spex, Jobin Yvon Inc., France). Data acquisition was performed in buffer B, using a 115F-QS quartz cuvette (Hellma GmbH & Co. KG, Germany) with an excitation wavelength λEX ) 280 nm. Collected data were corrected for the buffer contribution. All measurements were performed at 25 °C.

Kapłon et al. SDS/Polyacrylamide Gel Electrophoresis. Proteins were separated on SDS/polyacrylamide gels according to Laemmli10 and stained with either Coomassie Brilliant Blue R 25014 or carbocyanine (Stains-All; Sigma, Poland) dye.15,16 Molecular mass protein standards from Fermentas (Lithuania) were used. Disorder Predictions. In silico analysis of the naturally disordered regionswerecarriedoutusingaPONDRVL-XT(http://www.pondr.com),17,18 and DISOPRED 2 (http://bioinf.cs.ucl.ac.uk),19 neural network predictors with the default settings and the full-length Stm sequence from the NCBI Database (NP_942112).

Results Expression and Purification. The full-length Stm, Cterminally tagged with an affinity tag consisting of six histidine residues (Stm-His, Figure 1A) was expressed in E. coli. We tried to use Coomassie Brilliant Blue R-250 stained SDS/ polyacrylamide gels in our preliminary experiments of the expression analysis. Unfortunately, it was very hard to observe any expression of Stm-His using this detection method. It is a well-known fact that binding Coomassie Brilliant Blue R-250 dye depends on a positively charged residue with adjoining hydrophobic residues.20 This was probably the reason why Stm, a protein of extremely low hydrophobicity, exhibited lowered Commassie-binding. As indicated in the introduction, Stm is extremely hydrophilic; in total, 35% of Stm is composed of acidic residues.4 Therefore, to detect Stm, we decided to use carbocyanine (Stains-All). The Stains-All dye stains blue acidic and calcium-binding proteins, whereas all other proteins are stained red.16 As shown in Figure 1B, Stm-His, as well as other derivatives of Stm described below, are easily stained by the Stains-All dye. In particular, for Stm-His, two blue bands, not observed before the IPTG induction, could be detected (Figure 1B, compare lanes 3 and 2). At its N-terminus, the Stm-His protein contains a 20-residue long fragment, which was predicted in silico to be a signal peptide (SP).4 This is justified insofar as Stm is active in the extracellular compartment, which is an otic cavity6 and, thus, most probably is devoid of SP. Because of this, we assumed that the observed heterogeneity of Stm-His may reflect incomplete digestion of SP during the export of StmHis to the bacterial periplasma. Therefore, in our further experiments, we constructed, expressed, and studied derivatives of Stm, which were completely devoid of the SP sequence. The truncation of SP apparently stabilized the recombinant Stm, because for all subsequently studied Stm derivatives (Figure 1A), only a single blue-stained band with a mobility corresponding to the expected molecular mass could be observed (Figure 1B, lanes 5, 7, and 9). Initially, we decided to obtain an SP-devoid recombinant Stm, tagged with eight His residues at the N-terminus (His-Stm∆SP, Figure 1A). Surprisingly, preliminary purification experiments using a Talon-Co2+ affinity resin showed that, under the standard conditions recommended by the manufacturer (Clontech, U.S.A.), most of the HisStm∆SP protein remained unbound by this resin (data not shown). This could be due to either proteolytic digestion or the hiding of the N-terminal region of His-Stm∆SP, which would preclude interaction with Co2+ ions. And so the Stm derivative with eight His residues placed at the C-terminus of the recombinant protein was produced. Simultaneously, a nineamino acid peptide (Strep-tag II,21), which had been developed as an affinity tool for the purification of corresponding fusion proteins with streptavidin columns (Strep-Tactin,21), was attached to the N-terminus (Strep-Stm∆SP-His, Figure 1A). It is generally accepted that a double-tag strategy facilitates the purification of full-length proteins and increases protein purity

Starmaker Exhibits Properties of Disordered Protein

due to two independent and subsequent purification steps. Yet again, purification experiments carried out according to standard protocols resulted in a very poor yield of the double-tagged Stm. Approximately 100 µg of Strep-Stm∆SP-His was obtained from 1 L of bacterial culture (data not shown). Because ESI mass spectrometry analyses showed that both tags were present in the purified Strep-Stm∆SP-His (data not shown and also see below), we assumed that for some reason the structure of Stm prevents tags from interacting with appropriate resins and, thus, precludes using an affinity chromatography technique based on tags placed either on the C- or N-termini of Stm. Therefore, our next purification strategy was based on standard protein fractionation techniques and the nontagged, SP-devoid Stm (Stm∆SP, Figure 1A) was expressed (see Figure 1B, lane 9). To clarify the bacterial lysate and to remove contaminating proteases, ammonium sulfate fractionation was applied as the first step of the purification procedure. Small-scale pilot experiments revealed that Stm∆SP is soluble in a fraction of ammonium sulfate saturated to 55%, whereas increasing the ammonium sulfate saturation to 75% led to the almost complete precipitation of Stm∆SP (data not shown). Thus, we decided to use 55-75% ammonium sulfate lysate cut (Figure 2C,D, compare lanes 5 and 6) as starting material. Because the primary structure analyses of Stm suggested that this protein might reveal characteristics of an intrinsically disordered protein (IDP; see below), size-exclusion chromatography was chosen as the next step of the purification procedure. According to published data, IDPs exhibit a substantially increased hydrodynamic volume relative to globular proteins, leading to a significant increase in their apparent molecular mass. The molecular mass of Stm and its derivatives, calculated on the basis of their amino acid composition, is about 65 kDa. However, as a putative IDP family member, Stm was expected to elute earlier as a protein with an apparently higher molecular mass, due to the increased hydrodynamic volume relative to the globular protein of the same molecular mass.22,23 Indeed, as shown on Figure 2A, Stm∆SP was eluted in a volume close to the void volume (V0) of the HiLoad 16/60 Superdex 200 pg column, and consequently, it was separated from many of the contaminating proteins present in the ammonium sulfate fraction (Figure 2A, inset, also compare lanes 6 and 7 in Figure 2C). As was shown by So¨llner et al.,4 Stm plays a crucial role in the biomineralization of otoliths, which are mostly composed of calcium carbonate. It has been suggested that repeating a series of alternating S and D residues in Stm could serve as a template for binding calcium ions. Hydroxyapatite, analogically as calcium carbonate, should be effectively bound by Stm. Accordingly, chromatography on hydroxyapatite was selected as the next step of purification. Because of the acidic character of Stm, the surface of the hydroxyapatite was loaded with calcium ions.24–26 Figure 2B illustrates the hydroxyapatite chromatography carried out according to the protocol especially elaborated for Stm in our laboratory. An appropriate stepwise gradient of phosphate ions allows effective separation of Stm∆SP from contaminating proteins. The purified Stm∆SP appeared as a single band on the 15% SDS/polyacrylamide gel (Figure 2C and D, lane 8). The densitometric analysis of the Coomassie Brilliant Blue R-250 stained gel revealed the purity of Stm∆SP at 94%. It is noteworthy that this value has been underestimated because, as discussed above, Stm∆SP poorly binds Coomassie Brilliant Blue R-250. The final preparation of Stm∆SP was also devoid of significant amounts of truncated Stm∆SP, as judged by in-gel Stains-All detection (Figure 2D, lane 8). To confirm the identity of Stm∆SP, ESI mass


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Figure 2. Purification of Stm∆SP. (A) Preparative gel filtration of Stm∆SP. The 55-75% (NH4)2SO4 fraction was obtained from a 2 L culture of E. coli BL21(DE3)pLysS cells transformed with the appropriate derivative of the pQE-80L expression vector (see Materials and Methods). After dialysis, the resulting solution, which was concentrated to a volume of 1 mL, was loaded onto the HiLoad 16/60 Superdex 200 pg ¨ KTAexplorer column equilibrated with buffer A and connected to the A (Amersham Biosciences) system. Fractions of 1 mL were eluted at a flow rate of 0.5 mL/min and subsequently analyzed by Coomassie Brilliant Blue R 250- stained 10% SDS/polyacrylamide gel (inset, each lane corresponds to the respective fraction, the arrow shows the recombinant Stm∆SP). Fractions 3-10, marked as hatched areas on the chromatogram, were combined, dialyzed against buffer B, and applied to the next step of the purification procedure (Figure 2B). (B) Hydroxyapatite chromatography. The sample (10 mL) from the HiLoad 16/60 Superdex 200 pg column was applied onto a HR5/5 column filed with 1 mL of HTP resin equilibrated with buffer B and connected to the A¨KTAexplorer system. The flow rate was 0.25 mL/min and 0.5 mL fractions were collected. Note that Stm∆SP is devoid of W and Y residues and, hence, it reveals no significant absorption at 280 nm. Taking this into consideration, absorption at 220 was also monitored during the separation process. The experimentally elaborated step gradient of phosphate was used to elute bound proteins. The hatched area indicates fractions containing pure Stm∆SP as judged by subsequent SDS/polyacrylamide gel electrophoresis (see Figure 2C,D). (C) Representative Coomassie Brilliant Blue R 250, stained SDS/polyacrylamide gel analysis of the expression and purification procedure of Stm∆SP. Lane 1, molecular mass standards; lanes 2 and 3, the whole bacterial cell extract before the induction of Stm∆SP with IPTG (TOT NI) and 3 h later (TOT 3 h; ca. 30 µg); lane 4, soluble protein fraction obtained after cell lysis (ca. 40 µg); lanes 5 and 6, proteins from 0-55% and 55-75% (NH4)2SO4 fractions, respectively (ca. 20 µg in lane 5 and 40 µg in lane 6); lane 7, the combined gel filtration fractions 3-10 (Figure 2A, hatched area; ca. 6 µg); lane 8, the purified Stm∆SP after hydroxyapatite chromatography (ca. 6 µg). (D) The same analysis as in (C), but the SDS/polyacrylamide gel was stained using Stains-All dye. Note that amounts of the applied samples were halved. In (C) and (D), the position of molecular mass standards in kDa are indicated on the left, and the arrows on the right mark positions of Stm∆SP.

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spectrometry was applied, resulting in a value of 64497.3 ( 4 Da, which differs from the expected value (64628.2 Da) by 131 mass units. The observed difference was caused by the specific digestion of N-terminal formylmethionine, as judged by the N-terminal protein sequence analysis (data not shown). The elaborated purification procedure typically yielded about 0.85 mg of Stm∆SP from 1 L of the culture medium. Moreover, this procedure seems to be universal for various Stm derivatives because it also allowed us to purify Strep-Stm∆SP-His with a similar yield and purity (data not shown). It is noteworthy that the ESI mass spectrometry analysis of the purified StrepStm∆SP-His confirmed the presence of both peptide tags (data not shown). Stm Exhibits Properties of an Intrinsically Disordered Protein. Both the extremely highly acidic character of Stm, which is probably crucial for its functioning as a calcium ionbinding protein,4 as well as the unusual properties observed during elaboration of the purification procedure, inspired us to take a closer look at the amino acid composition of Stm. To that end, Composition Profiler was used,27 a web-based tool for semiautomatic analysis of the enrichment or depletion of amino acids. Figure 3A shows the composition profiles of Stm and Stm∆SP residues in comparison to the PDB Select 25 data set, based on a subset of structures from the Protein Data Bank, biased toward the composition of proteins easy to crystallize, that is, by definition, proteins possessing an ordered structure. Our analysis revealed that both full-length Stm and Stm devoid of the SP sequence (Stm∆SP), thus better corresponding to the functional protein, have a distinctive amino acid composition. Generally, they are rich in polar and charged amino acids (E, D, S, K, T, and H) and simultaneously poor in nonpolar residues (V, L, I, F, M); some of them (W, Y, C) are even absent in both proteins. According to Dunker et al.,29 such an amino acid composition is probably a symptom of an intrinsic disorder, a distinguishing feature of intrinsically disordered proteins (IDPs), also known as intrinsically unstructured or natively unstructured proteins. Intrinsic disorder is essential for IDPs as their various biologically important functions stem either directly from the structural disorder state or from local folding/ordering in molecular recognition.23,28 Indeed, a more detailed analysis of the data presented in Figure 3A based on the classification elaborated by Dunker et al.29 indicates that, among polar and charged amino acids, three (S, E, K) can be categorized as disorder-promoting residues and the other three (D, T, H) as disorder-neutral residues. At the same time, the most seriously depleted nonpolar residues are either order-promoting (V, L, I, F) or disorder-neutral (M) residues. To determine whether the sequence attributes of Stm and Stm∆SP are similar to those of IDPs, we also analyzed the fractional difference between IDP compositions (DisProt 3.4 data set,30) and PDB S25 compositions. A comparison of these results with data obtained for Stm and Stm∆SP (Figure 3A) indicates that the overall trend for IDPs and Stm data sets is similar. Remarkably, the frequencies of five disorder-promoting residues (A, R, Q, G, and P) are significantly lower than in the data set for IDPs. At the same time, Stm and Stm∆SP are significantly rich in some other disorder-promoting residues (S, E, K) and considerably deficient in order-promoting residues (V, L, I, F). Three order-promoting residues (W, C, Y) are completely missing from the Stm proteins. All these results together suggest that Stm and its recombinant derivatives belong to the class of IDPs. This supposition is further supported by the Uversky plot (Figure 3B),22,29 which unquestionably classifies Stm and Stm∆SP as members of the IDP family. The data presented in Figure 3C

Kapłon et al.

were obtained using DISOPRED19 and PONDR VL-XT,17,18,31 predictors, two of the algorithms that are widely used for the prediction of disordered regions within amino acid sequences. The data clearly indicate that Stm and Stm∆SP are highly disordered, although a few potential regions of order were predicted to exist within their sequences. In particular, one region corresponding to the SP sequence (positions 2-22) was predicted by both algorithms to possess an ordered structure in the full-length Stm (Figure 3C). Interestingly, according to PONDR VL-XT, a few potential regions of order were predicted, two of them around residues 33-53 and 348-359 in the wild-type Stm and in Stm∆SP. Unfortunately, because the DISOPRED classifies these regions to be ordered, their actual character is ambiguous and needs to be experimentally studied. Low mean hydrophobicity and a high net charge associated with a disordered state preclude the formation of the hydrophobic cluster and promote an extended conformation by electrostatic repulsion. Consequently, IDPs are characterized by a substantially larger hydrodynamic volume relative to globular proteins, leading to an increase in their apparent molecular mass.22,23 The first experimental hint that Stm might reveal such characteristics was its atypical behavior during preparative gel filtration (Figure 2A). However, because of contaminants still present during preparative gel filtration, which may affect the observed elution volume, purified Stm∆SP was used to accurately determine its Stokes radius using a gel filtration technique. As shown in Figure 4, Stm∆SP was eluted from the Superdex 200 10/300 GL column as a single peak, with an elution volume corresponding to a Stokes radius of 78.6 ( 3 Å. This value is substantially higher than the 35.5 Å, the value which should be observed assuming there is a globular shape for the Stm∆SP molecule with a calculated molecular mass of 64497.0 Da. Obviously, the high value of the estimated Stokes radius might also result from the oligomerization of globular Stm∆SP. The symmetry of the Stm∆SP peak, the absence of any additional peaks resulting from the dissociation of the putative Stm oligomer, and finally, the protein concentrationindependence of the observed elution volume (Figure 4) all suggest that, at least under conditions applied in this study, Stm∆SP occurs as a monomer with a substantially larger hydrodynamic volume. As can be inferred from the amino acid composition, one of the reasons for the increased hydrodynamic volume of Stm may be its low hydrophobicity, resulting in a lack of the hydrophobic core. To acquire evidence for this supposition we analyzed the W residue fluorescence emission spectrum, which is widely used as a tool to make inferences regarding the local structure and dynamics of proteins. Because the wild-type Stm and Stm∆SP derivative does not contain any W residue, the purified Strep-Stm∆SP-His protein, which has one W residue within the Strep-tag II sequence, was used. Figure 5A presents the fluorescence emission spectrum of the StrepStm∆SP-His measured with an excitation of 280 nm. The emission spectrum has a maximum fluorescence (λmax) of 350 nm, suggesting the occurrence of the W residue in a polar environment. This suggests that it is unlikely that Stm possesses an extensive hydrophobic core, otherwise, the W residue would probably be engaged in its formation. However, this experiment cannot exclude the existence of hydrophobic clusters, which might be created without the participation of the N-terminal W residue. The presence of some residual hydrophobic clusters might not interfere with the extended conformation of Stm. However, further experiments are needed to confirm such assumptions.



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Figure 4. High value of the Stokes radius of Stm∆SP suggests its low compactness. Analytical gel filtrations were performed on a Superdex 200 10/300 GL column with a flow rate of 0.5 mL/min and an injection volume of 0.1 mL. The column was equilibrated with buffer B and the protein concentration of the samples was 1.57 mg/mL (solid line), 0.157 mg/mL (dashed line), and 0.0157 mg/mL (short dashed line). Based on the calibration curve (inset), Stm∆SP can be characterized as a protein with an apparent Stokes radius of 78.6 ( 3 Å. Note that this value did not depend on the protein concentration. In the inset, the standard proteins are shown as open triangles and Stm∆SP is shown as a black circle.

Figure 3. Computational analysis of the Stm sequence. (A) Amino acid composition of Stm. The amino acid composition of Stm, Stm∆SP, and IDPs (represented by the DisProt 3.4 data set,30) are shown relative to the ordered globular proteins PDB S25 data set.27 Analysis was accomplished using the Composition Profiler.27 Values below zero indicate depletion in a given residue, whereas positive values indicate the wealth of a given residue. Note that for W, C, and Y residues the largest possible magnitude (-1) for a negative peak was observed for Stm and Stm∆SP, indicating that W, C, and Y are completely missing from these proteins. The residue order is based on the B-factor,43 which estimates the flexibility of each residue with the most rigid residues on the left and the most flexible on the right. (B) Charge-hydropathy analysis - Uversky plot.22 A PONDR (http://www.pondr.com.) server was used for the analysis of the mean hydrophobicity and net charge of the wild-type Stm and the Stm∆SP recombinant derivative. The results were compared with values obtained for proteins used originally by Uversky et al.44 The solid line represents the border between IDP family classified proteins (open triangles) and globular proteins (solid triangles). (C) The prediction of disordered regions from an amino acid sequence. The prediction of the degree of disorder in wild-type Stm was calculated from its primary structure using PONDR VL-XT (dashed line) and DISOPRED 2 (solid line) neural network predictors. For the PONDR predictor the score of >0.5 (horizontal dashed line) indicated a high probability of disorder, whereas the boundary value for DISOPRED 2 was a score of 0.05 (horizontal solid line). Note, that the same predictions for Stm∆SP yielded an essentially identical plot. The only difference was observed at the N terminus region, because Stm∆SP is devoid in 21 amino acids of the SP sequence (not shown).

Figure 5. Spectroscopic analyses of the Stm. (A) The fluorescence emission spectrum of Strep-Stm∆SP-His. The excitation wavelength was 280 nm and the protein concentration was 0.3 µg/µL. Data acquisition was performed in buffer B at 25 °C. (B) CD spectrum of Stm∆SP. The far-UV CD spectrum of Stm∆SP was recorded in buffer B in 0, 1, and 5 M GdmCl. The protein concentration was 2.15 µM.

The far-UV CD spectrum of Stm∆SP is typical for a disordered protein, as can be seen from its large negative ellipticity at 198 nm (Figure 5B). However, the observed ellipticity values at 200 and 222 nm suggest the existence of some residual secondary structures. To acquire further evidence for the occurrence of a secondary structure within Stm∆SP, we

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recorded the CD spectra in the presence of guanidium chloride (GdmCl) at 1 and 5 M concentrations. The comparison of these spectra (Figure 5B) with the spectrum recorded in the absence of the denaturant indicated that ellipticity at 220 nm increases, reflecting the denaturation of secondary structures. The data presented above, taken together, strongly suggest that full-length Stm and the recombinant Stm∆SP derivative belong to a class of IDPs.

Discussion Calcium carbonate biominerals are one of the most abundant minerals on the surface of the Earth.32 In eukaryotes, all biologically controlled calcium carbonate minerals are associated with an organic matrix, which represents only a small fraction (from 0.1 wt % to a few wt %) of the total biomineral. The matrix plays an essential role in mineralization, and its functions are crystal nucleation, the control of crystal shape, and crystal growth and inhibition.32 Among the proteinaceous moiety, the key components of the matrix are unusually acidic proteins, rich in aspartic acid residues. Several acidic proteins have been identified, but none of them have been established in polymorph selection, possibly due to the difficulty of working with highly acidic proteins.33 Stm, which also can be classified as a very acidic protein (24.6% of D and 10.6% of E residues), was the first protein shown capable of influencing the process of biomineralization. To lay the foundation for systematic studies on the molecular mechanism of Stm-dependent calcium carbonate biomineralization, we aimed at elaborating experimental conditions under which pure recombinant Stm could be obtained with a satisfying yield. Because initial experiments demonstrated that full-length Stm is prone to degradation, possibly due to the presence of the SP sequence, we decided to express Stm as a protein devoid of SP. Two Stm recombinants (His-Stm∆SP and Strep-Stm∆SPHis) were subjected to purification via affinity chromatography under standard conditions. Surprisingly, only a small fraction of the recombinant Stm molecules could be effectively bound by the affinity resins, despite the fact that mass spectrometry analyses unambiguously confirmed the presence of the appropriate affinity tags. It is not clear why the tags are not able to interact with affinity resins. One reasonable explanation for these unexpected observations could be the involvement of the Stm C- and N-termini in some interaction with other parts of the Stm molecule or with other Stm molecules. The latter, very attractive explanation, could indicate that Stm exists in an oligomeric state. Unfortunately, at the moment, we do not have experimental data supporting this hypothesis. Finally, the purification procedure based on standard protein fractionation techniques was elaborated for the nontagged Stm (Stm∆SP), with the amino acid sequence corresponding to the sequence of mature Stm, that is, devoid of the SP sequence.4 The protein can be obtained with a reasonable yield and purity. This lays the foundation for the systematic biochemical characterization of the Stm∆SP molecule. Because our procedure results in a protein that completely lacks post-translational modifications, one of the future tasks could be analysis of the possible role of post-translational modifications on Stm∆SP structure and function. The post-translational modifications are believed to exert a significant effect on the structure of DSPP, a human homologue of Stm.34 Many of the observations and analyses presented above indicate that Stm∆SP appears to have properties characteristic of IDPs, a class of proteins that lack, at least in vitro under

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physiological conditions, rigid tertiary structure, and exist instead as highly dynamic ensembles of interconverting structures.22,23,29 IDPs carry out numerous important biological functions, which are invariably associated with their structural disorder. Recently, it has been suggested that IDPs actually fall into five broad functional classes based on their mode of action.35 The first class is that of an entropic chain, with functions stemming directly from protein disorder. IDPs in the other four categories, including the so-called effectors, scavengers, assemblers, and display sites, function via molecular recognition. The major functional asset of these IDPs is related to their significant disorder-order transition, that is, induced local folding upon binding to the respective target. Interestingly, due to their highly flexible structure, IDPs or proteins having disordered regions are typically involved in biological processes in which interactions with multiple partners are often involved.36,37 As noted recently, many proteins involved in biomineralization exhibit structural trends toward extended, random coil, or other unstable structures and, thus, can be classified as IDPs.38 Which of the putative partners of Stm are able to induce a disorder-order transition? Some very recent data coming from studies on otolith formation suggest possible candidates. The process of otolith formation can be separated into two stages. During the first stage seeding particles composed of glycogen and the otolith matrix protein-1 (OMP-1) form the nucleus of the otolith.8,39,40 Then the otoliths grow diurnally, resulting in the formation of daily rings within their microstructure.41 A ring is composed of an incremental zone predominated by a calcium carbonate and a discontinuous zone comprising the organic matrix.42 So¨llner et al.4 have demonstrated that successive rings were no longer present when Stm expression was blocked using antisense oligonucleotides. Simultaneously, a change in the crystal lattice and, thus, a change in otolith morphology was observed. In the most severely affected otoliths, which were devoid of Stm, the organic matrix did not run in radial directions. Instead, the organic matrix was excluded from a single, large inorganic crystal. A simple interpretation of these findings is that Stm is an indispensable component of the matrix involved in its organization, most likely upon encountering a proper proteinaceous partner. Very recently, immunohistological analysis indicated that layers in the organic matrix consist of two proteins, OMP-1, and otolin-1. Because OMP-1 and otolin-1 are localized at almost the same positions, it has been suggested that these proteins, which are necessary for normal otolith growth and their correct anchoring onto the sensory macule, are also likely to interact with each other in vivo.39,40 Immunochemistry has also shown that Stm is an integral component of otoliths throughout all stages of otolith development.6 Thus, it is possible that all three proteins participate in a complex set of interactions that would represent the mechanistic foundation for proper otolith formation and calcification. Acknowledgment. We thank professor Teresa Nicolson (Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland, OR 97201) for the supply of the Starmaker cDNA, cloned into pCR4-TOPO plasmid. This work was supported by the Polish Ministry of Science and Higher Education Grant 2827/P01/2007/32 and by the Wrocław University of Technology.

References and Notes (1) Ross, M. D.; Pote, K. G. Some properties of otoconia. Philos. Trans. R. Soc. London, Ser. B 1984, 304 (1121), 445–452. (2) Xiao, S.; Yu, C.; Chou, X.; Yuan, W.; Wang, Y.; Bu, L.; Fu, G.; Qian, M.; Yang, J.; Shi, Y.; Hu, L.; Han, B.; Wang, Z.; Huang, W.;


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BM800135M

Starmaker Exhibits Properties of an Intrinsically Disordered Protein

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